Sex differences in brain and behavior exist across vertebrates. Several of these models have become powerful tools for elucidating mechanisms regulating development of neural structure and function, because they allow controlled investigation of naturally occurring differences among individuals that are otherwise highly similar. This strategy requires the careful selection of model systems with key advantages for particular questions and hypotheses. The zebra finch song system is ideally suited to study the molecular basis of development of neural structure and function for many reasons, including one of the largest sex differences in any tractable model system and remarkable similarity to mammals, including humans, in the circuitry regulating social behaviors. Experiments in this proposal will test a novel hypothesis, that estradiol (E2) interacts with male-typical levels of sex chromosome gene expression to masculinize structure and function of the neural song circuit. While the roles of sex steroids in development of neural circuitry are well established for many model systems, interactions between these hormones and other molecules important for differentiation of brain regions controlling social behaviors are largely unknown. The proposed studies are based on three sex chromosome genes: Secretory carrier membrane protein 1 (SCAMP1), sorting nexin 2 (SNX2), and tyrosine kinase B (the high affinity receptor for brain derived neurotrophic factor, BDNF). Expression of each of them is increased in song control nuclei of developing males compared to females. Specific Aim 1 will test the hypothesis that E2 increases expression of these genes or the proteins they encode within developing song control nuclei using in situ hybridization and immunonistochemistry. Aim 2 will test the hypothesis that expression of the genes enhances responsiveness of the song system to E2. Hatchling females will be treated with E2, and at specific stages of development important for morphological differentiation and song learning, siRNA will be used to individually inhibit expression of SCAMP1, SNX2 and trkB. Parallel studies will be done in males, treated with an estrogen synthesis inhibitor. Aim 3 will evaluate a set of mechanistic hypotheses regarding the roles of E2, norepinephrine (NE), BDNF, and the three sex chromosome genes (trkB, SCAMP1 and SNX2). Specifically, we will test the ideas that the ligands NE and/or BDNF are increased by E2 during song system differentiation using manipulations parallel to those in Specific Aim 1. We will also determine effects of NE and BDNF on morphological development and song learning by inhibiting them during appropriate developmental stages. A third study will use siRNA to inhibit SCAMP1 to test whether it facilitates release of NE into song nuclei. Finally, siRNA followed by autoradiography will be employed to determine whether SNX2 enhances the ability of BDNF and/or NE to act in the song system by increasing the availability of membrane receptors. PUBLIC HEALTH RELEVANCE: The proposed work is particularly relevant to the mission of NIMH, and will elucidate factors associated with development of a neural circuit with a clear set of behavioral functions -- the learning and production of a primary means of social interaction, vocal communication. The studies will determine mechanisms by which genes and their products influence the maturation of the structure and function of this neural circuit, focusing on the potential for interaction with steroid hormones and neurotransmitters. The experiments will therefore increase the understanding of factors critical to normal development of brain morphology and behavior, and can lead to the discovery of solutions to problems, including those associated with mental health disorders such as autism, schizophrenia and depression.